The innate immune system detects the presence of invading microorganisms through a panel of pattern recognition receptors. These receptors can sense the presence of intracellular or extracellular microbial patterns such as DNA, RNA, and bacterial cell wall components. Activation of signaling pathways downstream of ligand binding leads to the induction of pro-inflammatory cytokines such as type I Interferons (IFN). While the induction of IFN is typically considered an anti-viral mechanism, bacterial pathogens also induce type I interferon during infection. Interestingly, the mechanisms underlying IFN induction during infection are largely unknown. In general, we are interested in mechanisms by which recognition of bacteria by the innate immune results in the induction of IFN. Our specific interest focuses on cyclic-di-GMP (c-di-GMP), a bacterial signaling molecule that regulates biofilm formation, motility and virulence. This molecule has recently been proposed to induce a type I interferon response following translocation into host cells. However, the direct host intracellular molecular sensor has yet to be identified. The proposed works seeks to confirm the cytosolic sensing of c-di-GMP, identify bacterial pathogens that induce IFN through cytosolic sensing of c-di-GMP, and identify the eukaryotic molecule(s) essential for cytosolic recognition of c-di-GMP and induction of IFN. C-di-GMP is a molecule produced exclusively by bacteria and eukaryotic organisms do not appear to possess the enzymes required for its synthesis. Therefore, this molecule represents a unique target for drug design. Research into understanding the mechanism of cytosolic detection of c-di-GMP will aid in generating better vaccines, adjuvants and therapies. PUBLIC HEALTH RELEVANCE: Bacterial pathogens use cyclic-di-GMP to regulate virulence factors. In addition, host cells sense this molecule during infection leading to a potent immune response against the bacteria. The goal of this application is to identify the essential host components required to respond to cyclic-di-GMP during infection. Understanding the innate immune system's response to cyclic-di-GMP is an essential aspect of designing effective vaccines against bacterial pathogens.